Sustained release formulation of a peptide

ABSTRACT

This invention is directed to a sustained release composition comprised of Compound (A) having the formula 
                         
or a pharmaceutically acceptable salt thereof, and a copolymer comprised of poly-(I)-lactic-glycolic-tartaric acid wherein the amino group of Compound (A) is ionically bound to a carboxyl group of the copolymer and wherein further the composition may be made into a sustained release pharmaceutical composition with pharmaceutically acceptable carrier(s).

This application is a 371 of PCT/US00/22464 filed on Aug. 16, 2000,which claims the benefit of 60/149,649 filed on Aug. 18, 1999.

BACKGROUND OF THE INVENTION

This invention pertains to a sustained release complex, Compound (I),which comprises Compound (A), having the formula

or a pharmaceutically acceptable salt thereof, and a copolymercomprising poly-(I)-lactic-glycolic-tartaric acid (P(I)LGT), wherein theamino group of said Compound (A) is ionically bound to a carboxyl groupof the P(I)LGT. The present invention further pertains to a process formaking said sustained release complex. Further still, the presentinvention is directed to a pharmaceutical composition comprising saidsustained release complex and a pharmaceutically acceptable carrier(s).

Further, since Compound (A) is an analogue of somatostatin and it iswell known to those skilled in the art that the known and potential usesof somatostatin are varied and multitudinous, this invention is alsodirected to the use of Compound (A), Compound (I) or microparticles ofCompound (I) to treat a disease or condition in a patient in needthereof, which comprises administering Compound (A), Compound (I) ormicroparticles of Compound (I) to said patient, wherein the diseases orconditions to be treated are selected from the group consisting ofgastroenterological conditions and/or diseases, such as Crohn's disease,systemic sclerosis, external and internal pancreatic pseudocysts andascites, VIPoma, nesidoblastosis, hyperinsulinism, gastrinoma,Zollinger-Ellison Syndrome, diarrhea, AIDS related diarrhea,chemotherapy related diarrhea, scleroderma, Irritable Bowel Syndrome,pancreatitis, upper gastrointestinal bleeding, postprandial portalvenous hypertension especially in cirrhotic patients, complications ofportal hypertension, small bowel obstruction, gastroesophageal reflux,duodenogastric reflux and in treating endocrinological diseases and/orconditions, such as Cushing's Syndrome, gonadotropinoma,hyperparathyroidism, Graves' Disease, diabetic neuropathy, maculardegeneration, hypercalcemia of malignancy, Paget's disease, andpolycystic ovary disease; in treating various types of cancer such asthyroid cancer, leukemia, meningioma and conditions associated withcancer such as cancer cachexia; in the treatment of such conditions ashypotension such as orthostatic hypotension and postprandial hypotensionand panic attacks.

Many drug delivery systems have been developed, tested and utilized forthe controlled in vivo release of pharmaceutical compositions. Forexample, polyesters such as poly(DL-lactic acid), poly(glycolic acid),poly(ε-caprolactone) and various other copolymers have been used torelease biologically active molecules such as progesterone; these havebeen in the form of microcapsules, films or rods (M. Chasin and R.Langer, editors, Biodegradable Polymers as Drug Delivery Systems,Dekker, NY 1990). Upon implantation of the polymer/therapeutic agentcomposition, for example, subcutaneously or intramuscularly, thetherapeutic agent is released over a specific period of time. Suchbio-compatible biodegradable polymeric systems are designed to permitthe entrapped therapeutic agent to diffuse from the polymer matrix. Uponrelease of the therapeutic agent, the polymer is degraded in vivo,obviating surgical removal of the implant. Although the factors thatcontribute to polymer degradation are not well understood, it isbelieved that such degradation for polyesters may be regulated by theaccessibility of ester linkages to non-enzymatic autocatalytichydrolysis of the polymeric components.

Several EPO publications and U.S. Patents have addressed issues ofpolymer matrix design and its role in regulating the rate and extent ofrelease of therapeutic agents in vivo.

For example, Deluca (EPO Publication 0 467 389 A2) describes a physicalinteraction between a hydrophobic biodegradable polymer and a protein orpolypeptide. The composition formed was a mixture of a therapeutic agentand a hydrophobic polymer that sustained its diffusional release fromthe matrix after introduction into a subject.

Hutchinson (U.S. Pat. No. 4,767,628) controlled the release of atherapeutic agent by uniform dispersion in a polymeric device. It isdisclosed that this formulation provides for controlled continuousrelease by the overlap of two phases: first, a diffusion-dependentleaching of the drug from the surface of the formulation; and second,releasing by aqueous channels induced by degradation of the polymer.

PCT publication WO 93/24150 discloses a sustained release formulationcomprising a peptide having a basic group and a carboxy-terminatedpolyester.

U.S. Pat. No. 5,612,052 describes cation-exchanging microparticles madetypically of carboxyl-bearing polyester chains onto which basicbioactive agents are immobilized to provide a control release systemwithin an absorbable gel-forming liquid polyester.

Compound (A) is described and claimed in U.S. Pat. No. 5,552,520, whichis assigned to the assignee hereof.

PCT publication WO 97/40085, assigned to the assignee hereof, disclosesbiodegradable polyesters comprising lactic acid units, glycolic acidunits and hydroxy-polycarboxylic acid units such as tartaric acid orpamoic acid and processes for making said polyesters. More specifically,it discloses poly-lactide-glycolide-tartaric acid polymers in the ratio65/33/2, respectively.

PCT publication WO 94/15587, assigned to the assignee hereof, disclosesionic conjugates of polyesters having free COOH groups with a bioactivepeptide having at least one effective ionogenic amine. Morespecifically, it discloses that the polymers are made polycarboxylic byreacting the co-polymers with malic acid or citric acid. U.S. Pat. No.5,672,659, is the U.S. national phase continuation application of WO94/15587. U.S. Pat. No. 5,863,985 is a continuation of U.S. Pat. No.5,672,659. Pending U.S. application Ser. No. 09/237,405 is a CIP of U.S.Pat. No. 5,863,985, which additionally discloses a polyester which mustinclude citric acid, ε-caprolactone and glycolide; compositionscomprising the immediately foregoing polyesters and a polypeptide; apolyester that must include tartaric acid as one of its members;compositions comprising the immediately foregoing polyester and apolypeptide; and the foregoing compositions in the shape of rods whichare optionally coated with a biodegradable polymer.

PCT publication WO 97/39738, assigned to the assignee hereof, disclosesa method of making microparticles of a sustained release ionic conjugateas described in WO 94/15587.

The contents of the foregoing patents, applications and publications areincorporated herein in their entirety.

The present invention is directed to a preferred embodiment of asustained release ionic conjugate of polymerpoly-lactide-glycolide-tartaric acid and Compound (A), also known asCompound (I), which is characterized by the surprising and non-obviousproperty of zero-order release of Compound (A) from the conjugate. Morepreferably, the ionic conjugate, Compound (I), is in the form ofmicroparticles.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Shows the in vivo release profile of Compound (A) from a sampleof Compound (I) in dog, wherein the sample of Compound (I) consists ofabout 11.23% Compound (A), the polymer is I-lactide:glycolide:tartaricacid (72:27:1) and where Compound (I) is administered intramuscularly asmicroparticles. The irradiated sample refers to a sample of Compound (I)which was irradiated with γ-rays from a Cobalt source.

SUMMARY OF THE INVENTION

The present invention is directed to a Compound (I) which comprisesCompound (A), having the formula

and a polymer, wherein the polymer comprises lactide units, glycolideunits and tartaric acid units where the ratio in the polymer: of thelactide units is from and including about 71% to about 73%, of theglycolide units is from and including about 26% to about 28%; and of thetartaric acid units is from and including about 1% to about 3%; andwhere the amino group of Compound (A) is ionically bonded to acarboxylic group of the acid units of the polymer.

A preferred embodiment of Compound (I) is where the polymer consists ofabout 72% lactide units, about 27% glycolide units and about 1% tartaricacid units.

A preferred embodiment of the immediately foregoing Compound (I) iswhere the percentage of Compound (A) in Compound (I) is about 8% toabout 12%.

In another aspect, the present invention is directed to microparticlesof Compound (I) which comprises Compound (A), having the formula

and a polymer, wherein the polymer comprises lactide units, glycolideunits and tartaric acid units where the ratio in the polymer of thelactide units is from and including about 71% to about 73%, of theglycolide units is from and including about 26% to about 28%; and of thetartaric acid units is from and including about 1% to about 3%; andwhere the amino group of Compound (A) is ionically bonded to acarboxylic group of the acid units of the polymer.

Preferred microparticles of Compound (I), as described hereinabove, ofthis invention are those microparticles having a mean microparticle sizeof about 10 microns to about 100 microns.

More preferred microparticles of Compound (I), as described hereinabove,of this invention are those microparticles having a mean microparticlesize of about 40 microns to about 70 microns.

Even more preferred microparticles of the present invention is where themicroparticles exhibit a zero-order release profile of Compound (A) fromthe microparticles.

In yet another aspect, the present invention is directed to apharmaceutical composition comprising microparticles comprising Compound(I) which comprises Compound (A), having the formula:

and a polymer, wherein the polymer comprises lactide units, glycolideunits and tartaric acid units where the ratio in the polymer: of thelactide units is from and including about 71% to about 73%, of theglycolide units is from and including about 26% to about 28%; and of thetartaric acid units is from and including about 1% to about 3%; andwhere the amino group of Compound (A) is ionically bonded to acarboxylic group of the acid units of the polymer;and optionally a pharmaceutically acceptable carrier, diluent oradjuvant.

In a further aspect, the present invention is directed to a method oftreating a disease or condition in a patient in need thereof, whichcomprises administering to said patient an effective amount of Compound(A), as described hereinabove, or a pharmaceutically acceptable saltthereof, wherein the disease or condition is selected from the groupconsisting of systemic sclerosis, pancreatic pseudocysts, pancreaticascites, VIPoma, nesidoblastosis, hyperinsulinism, gastrinoma,Zollinger-Ellison Syndrome, hypersecretory diarrhea, scleroderma,irritable bowel syndrome, upper gastrointestinal bleeding, postprandialportal venous hypertension, complications of portal hypertension, smallbowel obstruction, duodenogastric reflux, Cushing's Syndrome,gonadotropinoma, hyperparathyroidism, diabetic neuropathy, maculardegeneration, hypercalcemia of malignancy, Paget's disease, meningioma,cancer cachexia, psoriasis, hypotension and panic attacks.

In an even further aspect, the present invention is directed to a methodof treating a disease or condition in a patient in need thereof, whichcomprises administering to said patient an effective amount of Compound(I), as described hereinabove, wherein the disease or condition isselected from the group consisting of systemic sclerosis, pancreaticpseudocysts, pancreatic ascites, VIPoma, nesidoblastosis,hyperinsulinism, gastrinoma, Zollinger-Ellison Syndrome, hypersecretorydiarrhea, scleroderma, irritable bowel syndrome, upper gastrointestinalbleeding, postprandial portal venous hypertension, complications ofportal hypertension, small bowel obstruction, duodenogastric reflux,Cushing's Syndrome, gonadotropinoma, hyperparathyroidism, diabeticneuropathy, macular degeneration, hypercalcemia of malignancy, Paget'sdisease, meningioma, cancer cachexia, psoriasis, hypotension and panicattacks.

In still a further aspect, the present invention is directed to a methodof treating a disease or condition in a patient in need thereof, whichcomprises administering to said patient an effective amount ofmicroparticles of Compound (I), as described hereinabove, wherein thedisease or condition is selected from the group consisting of systemicsclerosis, pancreatic pseudocysts, pancreatic ascites, VIPoma,nesidoblastosis, hyperinsulinism, gastrinoma, Zollinger-EllisonSyndrome, hypersecretory diarrhea, scleroderma, irritable bowelsyndrome, upper gastrointestinal bleeding, postprandial portal venoushypertension, complications of portal hypertension, small bowelobstruction, duodenogastric reflux, Cushing's Syndrome, gonadotropinoma,hyperparathyroidism, diabetic neuropathy, macular degeneration,hypercalcemia of malignancy, Paget's disease, meningioma, cancercachexia, psoriasis, hypotension and panic attacks.

DETAILED DESCRIPTION

The term “about” as used herein in association with parameters andamounts, means that the parameter or amount is within ±5% of the statedparameter or amount.

The term “microparticle(s)” as used herein, refers to the micron sizeparticles of the ionic conjugate comprising Compound (A) andpoly-lactide-glycolide-tartaric acid polymer, which are preferably inessentially spherical form.

The instant application denotes amino acids using the standard threeletter abbreviation known in the art, for example Phe=phenylalanine;Abu=α-aminobutyric acid.

As is well known to those skilled in the art, the known and potentialuses of somatostatin are varied and multitudinous. Somatostatin is knownto be useful in the treatment of the diseases and/or conditions listedhereinbelow. The varied uses of somatostatin may be summarized asfollows: Cushings Syndrome (see Clark, R. V. et al, Clin. Res. 38, p.943A, 1990); gonadotropinoma (see Ambrosi B., et al., Acta Endocr.(Copenh.) 122, 569–576, 1990); hyperparathyroidism (see Miller, D., etal., Canad. Med. Ass. J., Vol. 145, pp. 227–228, 1991); Paget's disease(see, Palmieri, G. M. A., et al., J. of Bone and Mineral Research, 7,(Suppl. 1), p. S240 (Abs. 591), 1992); VIPoma (see Koberstein, B., etal., Z. Gastroenterology, 28, 295–301, 1990 and Christensen, C., ActaChir. Scand. 155, 541–543, 1989); nesidioblastosis and hyperinsulinism(see Laron, Z., Israel J. Med. Sci., 26, No. 1, 1–2, 1990, Wilson, D.C., Irish J. Med. Sci., 158, No. 1, 31–32, 1989 and Micic, D., et al.,Digestion, 16, Suppl. 1.70. Abs. 193, 1990); gastrinoma (see Bauer, F.E., et al., Europ. J. Pharmacol., 183, 55 1990); Zollinger-EllisonSyndrome (see Mozell, E., et al., Surg. Gynec. Obstet., 170, 476–484,1990); hypersecretory diarrhea related to AIDS and other conditions (dueto AIDS, see Cello, J. P., et al., Gastroenterology, 98, No. 5, Part 2,Suppl., A163 1990; due to elevated gastrin-releasing peptide, seeAlhindawi, R., et al., Can. J. Surg., 33, 139–142, 1990; secondary tointestinal graft vs. host disease, see Bianco J. A., et al.,Transplantation, 49, 1194–1195, 1990; diarrhea associated withchemotherapy, see Petrelli, N., et al., Proc. Amer. Soc. Clin. Oncol.,Vol. 10, P 138, Abstr. No. 417 1991); irritable bowel syndrome (seeO'Donnell, L. J. D., et al., Aliment. Pharmacol. Therap., Vol. 4.,177–181, 1990); pancreatitis (see Tulassay, Z., et al.,Gastroenterology, 98, No. 5, Part 2, Suppl., A238, 1990); Crohn'sDisease (see Fedorak, R. N., et al., Can. J. Gastroenterology, 3, No. 2,53–57, 1989); systemic sclerosis (see Soudah, H., et al.,Gastroenterology, 98, No. 5, Part 2, Suppl., A129, 1990); thyroid cancer(see Modigliani, E., et al., Ann., Endocr. (Paris), 50, 483–488, 1989);psoriasis (see Camisa, C., et al., Cleveland Clinic J. Med., 57, No. 1,71–76, 1990); hypotension (see Hoeldtke, R. D., et al., Arch. Phys. Med.Rehabil., 69, 895–898, 1988 and Kooner, J. S., et al., Brit. J. Clin.Pharmacol., 28, 735P–736P, 1989); panic attacks (see Abelson, J. L., etal., Clin. Psychopharmacol., 10, 128–132, 1990); sclerodoma (see Soudah,H., et al., Clin. Res., Vol. 39, p. 303A, 1991); small bowel obstruction(see Nott, D. M., et al., Brit. J. Surg., Vol. 77, p. A691, 1990);gastroesophageal reflux (see Branch, M. S., et al., Gastroenterology,Vol. 100, No. 5, Part 2 Suppl., p. A425, 1991); duodenogastric reflux(see Hasler, W., et al., Gastroenterology, Vol. 100, No. 5, Part 2,Suppl., p. A448, 1991); Graves' Disease (see Chang, T. C., et al., Brit.Med. J., 304, p. 158, 1992); polycystic ovary disease (see Prelevic, G.M., et al., Metabolism Clinical and Experimental, 41, Suppl. 2, pp76–79, 1992); upper gastrointestinal bleeding (see Jenkins, S. A., etal., Gut., 33, pp. 404–407, 1992 and Arrigoni, A., et al., AmericanJournal of Gastroenterology, 87, p. 1131, (abs. 275), 1992); pancreaticpseudocysts and ascites (see Hartley, J. E., et al., J. Roy. Soc. Med.,85, pp. 107–108, 1992); leukemia (see Santini, et al., 78, (Suppl. 1),p. 429A (Abs. 1708), 1991); meningioma (see Koper, J. W., et al., J.Clin. Endocr. Metab., 74, pp. 543–547, 1992); and cancer cachexia (seeBartlett, D. L., et al., Surg. Forum., 42, pp. 14–16, 1991). Thecontents of the foregoing references are incorporated herein byreference.

Applicant has now discovered that Compound (A), which is a somatostatinagonist, Compound (I) and microparticles of Compound (I), areparticularly useful in treating the conditions, disorders and diseasesnoted hereinabove.

General Procedures:

Co-Polymer formation: The copolymer consisting of L-lactide, glycolideand L(+)-tartaric acid can be made according to methods well-known tothose skilled in the art and as enabled herein. Accordingly, a reactoris loaded with monomers of glycolide, L-lactide and L(+)-tartaric acidand stannous 2-ethyl hexanoate in toluene solution. Preferably the molarpercentages of L-lactide, glycolide, and L(+)-tartaric acid is about72/27/1, respectively.

The L(+)-tartaric acid is previously dried, preferably over silica gelin an Abderhalden drying apparatus for about 10 hours. The reactor isthen put under vacuum with stirring to remove toluene. The reactor,under an atmosphere of oxygen-free nitrogen, is then heated, preferablyby immersing it in an oil bath, temperature=about 180° C. to 190° C.,and stirring is increased to about 125 rpm. Prior to immersion, aheating tape is placed on the reactor lid. The time taken to completelymelt the reactor contents is noted, typically about 15 minutes for aload of about 300 g at about 180° C. Samples are taken every hour duringsynthesis and analyzed by GPC to determine the percentage residualmonomer and to obtain values for average molecular weight by number (Mn)and by weight (Mw) distributions. Typical reaction times are of theorder of about 9 to 15 hours. The final polymer is also analyzed bytitration to determine an acid number in meq/g and by GC to determineresidual unreacted monomer content. Further analyses include IR(detection of characteristic C═O peak); NMR (determination of lactideand glycolide content in polymer) and residual tin (determination ofresidual tin due to use of stannous 2-ethyl hexanoate as catalyst).

Purification/Sodium salt formation of the above copolymer: Residualmonomer (typically <5% (W/W)) is removed and the copolymer is convertedto it's sodium salt form (to promote ionic salt formation) in one step.The poly-L-lactic-co-glycolic-co-L(+)-tartaric acid copolymer (PLGTA) isdissolved in acetone by sonication in a sonication bath to give asolution with a concentration in the range of 19–21% PLGTA by weight.

To this solution is added a weak solution of an inorganic base such asNaOH or Na₂CO₃, preferably 0.2M sodium carbonate—Na₂CO₃ is used, in anamount so that the resulting concentration of sodium is 1 to 2 timesmolar excess, preferably 1.2 times molar excess, over copolymer carboxylgroups. The solution is left to stir for about 15 to 60 minutes,preferably 30 minutes, at room temp. to aid sodium salt formation. It isthen fed at about 50 to 300 ml/min, preferably about 100 ml/min, into ajacketed reactor containing de-ionized water cooled to about 1 to 4° C.,preferably 2.5° C., using a circulation bath; the amount of water isabout 20 to 30 times volumetric excess over acetone, preferably 20:1volumetric excess over acetone. The water is stirred at a ratesufficient to create surface turbulence in order to avoid polymeragglomeration during precipitation using a paddle linked to a stirrermotor.

Once precipitation is complete, the dispersion is left to stir for afurther 30 to 60 minutes to aid monomer removal before being placed incentrifuge bottles and spun. The supernatant is discarded and the cakesare resuspended in further de-ionized water, re-spun and dried,preferably by lyophilization.

Preparation of a Compound (A) Polymer Ionic Conjugate: The synthesisentails binding Compound (A) to the copolymer sodium salt in a medium inwhich both are soluble, preferably 3:1 (W/W) acetonitrile:water,followed by precipitation of the resulting ionic conjugate in de-ionizedwater and recovery of the water-insoluble conjugate precipitate formed.

A solution of the acetate salt of Compound (A) in de-ionized water isadded to a solution consisting of a washed Na salt of 12,000 MW 71/28/1to 73/26/1 PLGTA in acetonitrile (Range 24–26% (W/W) solution) to whicha weak base such as 0.5M Na₂CO₃ has been added so that it results inabout a 1.05 molar excess of Na over the acetate content of the Compound(A) acetate salt, and left to stir for about 5 minutes to provide analkaline environment, preferably pH 8, to neutralize Compound (A)'sacetate group. Approximate weight ratio of acetonitrile:water=3:1. Basedon target loading required (usually about 8% to about 12%), the quantityof Compound (A) required is determined. From this the volume of aqueoussodium carbonate required to neutralize the acetate of Compound (A) isdetermined and finally the volume of water for Compound (A) dissolutionis calculated based on a desired final acetonitrile:water (includingsodium carbonate added) volumetric ratio of about 3:1.

The Compound (A)-copolymer solution is left to stir for about 10 to 15mins. at about 0 to 5° C., preferably 2.5° C., to facilitate ionicbinding and discourage covalent binding (by use of low temperature)between the two components. The solution is then fed at a rate of about50 to 300 ml/min into about a 20–30 to 1 volumetric excess of de-ionizedwater over the volume of acetonitrile in the foregoing 3:1acetonitrile-water solution, stirred at a rate sufficient to providesurface agitation and avoid agglomeration and cooled to about 1 to 4°C., preferably 1.7° C., in a jacketed reactor connected to a circulationbath.

When precipitation is complete the dispersion is left to stir for afurther 30 to 60 minutes to aid removal of water-soluble Compound(A)-oligomer compounds (oligomers are those lower molecular weightfractions of PLGTA, which are undesirable since they are water soluble)before being placed in centrifuge bottles and spun at about 5000 rpm forabout 15 minutes in a centrifuge. The resultant centrifuge cakes areresuspended in de-ionized water and re-spun. They are then frozen anddried by lyophilization for 2 days and Compound (I) (Compound (A)ionically bound to PLGTA) is recovered. The loading is determined byHPLC analysis of the supernatant for unbound Compound (A) and nitrogenanalysis (the Compound (A) nitrogen content is known and the polymercontains no nitrogen whatsoever). Extraction of Compound (A) fromCompound (I) followed by HPLC analysis also allows determination ofloading.

Compound (I) Nebulization: In order to provide a formulation well-suitedfor injection into a patient, Compound (I) is formulated intomicrospheres by dissolving it in ethyl acetate and using ultrasonicatomization (a.k.a. nebulization) to spray the solution into coldtemperature, about −60° C. to −78° C., ethanol, isopropanol or a mixtureof hexane and isopropanol, preferably isopropanol, which results in theformation of microspheres of Compound (I) upon contact with the coldisopropanol. The Compound (I) ethyl acetate solution can be sterilizedby passing it through a 0.2 μm filter.

Compound (I) is dissolved in ethyl acetate preferably bysonication/stirring to give about 8% to about 12% (W/W) solution,preferably 12%, depending on polymer molecular weight and Compound (A)loading, both of which may after solution viscosity. This is fed atabout 4.90 ml/min. to 5.10 ml/min., preferably 5.00 ml/min. to anindustrial atomizer or nebulizer (Power—about 70%, Amplitude—about 80%,Frequency—about 34 to 35 kHz, preferably 34.50 kHz; in general thenebulizer should be powerful enough to generate a frequency which canuniformly spray (without “spitting”) the Compound (I) ethyl acetatesolution from about 8% to about 12% (W/W) in concentration, suchconcentrations lead to the formation of solid microspheres and thefrequency should be such that a mean particle size of between 40 micronsand 70 microns is obtained, which will allow ease of injection through a21-gauge or a 19-gauge needle) and nebulized into a volume ofisopropanol (IPA) that is 20 to 30 times, preferably 20 times,volumetric excess compared to the ethyl acetate volume, cooled to about−60° C. to about −78° C., cooling can be achieved, (e.g., via a reactorjacket, addition of dry ice or insertion of a cooling coil) and stirredat least at about 200 rpm (to avoid microsphere agglomeration).De-ionised water at a temperature of about 6° C. is fed at preferably1.5 L/min to the nebulizer jacket to eliminate any local heating effectswhich can cause fouling of the nebulizer tip due to ethyl acetateevaporation. The solution nebulized evenly and an off-white particulatedispersion is seen to form in the IPA. This is allowed to thaw to about0° C. to 22° C. over a period of about 30 mins. to 2 hrs before passingit through a 125 μm sieve (to remove any large non-injectabledroplets/particles) and on to a Whatman no.1 filter paper where it isvacuum-filtered. The filter cake is rinsed with further IPA and thenvacuum dried.

The present invention is illustrated by the following example but is notlimited by the details thereof.

EXAMPLE 1 Ionic Conjugate of P(I)LGTA (72/27/1) and Compound A

Step A: Synthesis of 300 g of P(I)LG/Tartaric Acid Copolymer(I-lactide:glycolide:tartaric Acid=72:27:1)

A reactor was loaded with monomers of glycolide (Purac Biochem,Netherlands, 68.71 g), lactide (Purac Biochem, Netherlands, 227.53 g)and L(+)-Tartaric acid (Riedel-de Haen, Seelze, Germany, article number33801, 3.75 g) and stannous 2-ethyl hexanoate (Sigma, St. Louis, Mo.,USA, article number S-3252) in toluene (Riedel-de Haen, Seelze, Germany)solution (0.0982M, 4.47 ml). This corresponded to molar percentages of71.81%; 26.82%; and 1.36% respectively of L-lactide, glycolide, andL(+)-tartaric acid.

The L(+)-tartaric acid was previously dried over silica gel (Riedel-deHaen, Seelze, Germany) in an Abderhalden drying apparatus for about 10hours. The reactor (connected to a pump via a liquid nitrogen trap) wasthen put under vacuum (0.04 mbar) with stirring for about 50 minutes toremove toluene. The reactor, under an atmosphere of oxygen-free nitrogen(BOC gases, Dublin, Ireland, moisture content of 8 VPM), was thenimmersed in an oil bath (Temperature=˜180° C.) and stirring wasincreased to 125 rpm. Prior to immersion, a heating tape (Thermolynetype 45500, input control setting=4) was placed on the reactor lid. Thetime taken to completely melt the reactor contents was noted, typicallyabout 15 minutes for a load of 300 g at about 180° C. Samples were takenevery hour during synthesis and analyzed by GPC to determine thepercentage residual monomer and to obtain values for average molecularweight by number (Mn) and by weight (Mw) distributions. Typical reactiontimes were of the order of about 15 hours. The final polymer was alsoanalyzed by titration to determine an acid number in meq/g and by GC todetermine residual unreacted monomer content. Further analyses includeIR (detection of characteristic C═O peak); NMR (determination of lactideand glycolide content in polymer) and residual tin (determination ofresidual tin due to use of stannous 2-ethyl hexanoate as catalyst).

Step B: Purification/Sodium Salt Formation with the Above Copolymer

Residual monomer (typically <5% (W/W)) was removed and the copolymer wasconverted to it's sodium salt form (to promote ionic salt formation) inone step. 81.05 g of a 12,000 g/mol 72/27/1poly-L-lactic-co-glycolic-co-L(+)-tartaric acid copolymer (acid numberby titration=0.231 meq/g) was dissolved in 324.24 g of acetone(Riedel-de Haen, Seelze, Germany) by sonication in a sonication bath(Branson, Danbury, Conn., USA) to give a solution with a concentrationof 20.00% PLGTA by weight.

To this solution was added 56.17 ml of 0.2M Na₂CO₃ (Aldrich, Gillingham,Dorset, UK), thus providing a 1.2 times molar excess of sodium overcopolymer carboxyl groups. The solution was left to stir for about 30minutes at room temp. to aid sodium salt formation. It was then fed at˜100 ml/min into a 10 L jacketed reactor containing 8.2 L of de-ionizedwater (approximately a 20:1 volumetric excess over acetone cooled toabout 2.5° C. using a circulation bath (Huber, Offenburg, Germany). Thiswater was stirred at 800 rpm to create surface turbulence and avoidpolymer agglomeration during precipitation using a paddle linked to astirrer motor.

Once precipitation was complete, the dispersion was left to stir for afurther 30 mins. to aid monomer removal before being placed incentrifuge bottles and spun at 5000 rpm for about 15 minutes in aSorvall centrifuge (DuPont Sorvall Products, Wilmington, Del., USA). Thesupernatant was discarded and the cakes were resuspended in furtherde-ionized water, respun and frozen in a freezer (−13° C.) overnightbefore being dried in a small-scale lyophilizer (Edwards, Crawley, WestSussex, UK) the next day. This lyophilizer contains no coolant system.After 5 days of lyophilization 65.37 g of washed copolymer wererecovered representing a yield of 80.65%.

Step C: Preparation of Compound (I)

A solution of 1.27 g of the acetate salt of Compound (A) (Batch 97K-8501from Kinerton Ltd., Dublin, Ireland, potency=85.8% (potency refers tothe percent free base peptide present in the peptide acetate salt);acetate=10.87%) in 5.87 g of de-ionized water was added to a solutionconsisting of 8.01 g of a washed Na salt of 12,000 MW 72/27/1 PLGTA in24.84 g acetonitrile (Riedel de-Haen) (24.38% (W/W) solution to which2.41 ml of 0.5M Na₂CO₃ (this corresponds to a 1.05 excess of Na over theacetate content of Compound (A)-acetate salt) had been added and left tostir for about 5 minutes to provide an alkaline environment (pH 8) forneutralization of Compound (A)'s acetate groups. Approximate weightratio of acetonitrile:water=3:1. Based on target loading required, thequantity of Compound (A) required was determined. From this the volumeof aqueous sodium carbonate required to neutralize the acetate ofCompound (A) was determined and finally the volume of water for Compound(A) dissolution was calculated based on a desired finalacetonitrile:water (including sodium carbonate added) volumetric ratioof 3:1.

The Compound (A)-copolymer solution was left to stir for about 15 mins.at about 2.5° C. to facilitate ionic and discourage covalent bindingbetween the two. The solution was then fed at ˜100 ml/min into 630 ml(approximately a 20:1 volumetric excess over acetonitrile) of de-ionizedwater stirred at 350 rpm (to provide surface agitation and avoidCompound (A)-copolymer agglomeration) and cooled to about 1.7° C. in a 6L jacketed reactor connected to a circulation bath.

When precipitation was complete the dispersion was left to stir for afurther 30 minutes to aid removal of water-soluble Compound (A)-oligomercompounds before being placed in centrifuge bottles and spun at 5000 rpmfor about 15 minutes in a Sorvall centrifuge (DuPont Sorvall Products,Wilmington, Del., USA). The resultant centrifuge cakes were resuspendedin de-ionized water and re-spun. They were then frozen and dried bylyophilization for 2 days. 8.30 g of the title product were recoveredrepresenting a yield of 91.38%. The loading was determined by HPLCanalysis of the supernatant for unbound Compound (A) and nitrogenanalysis (the Compound (A) nitrogen content is known and the polymercontains no nitrogen whatsoever). Extraction of Compound (A) fromCompound (I) followed by HPLC analysis also allows determination ofloading, which for this example was 11.25%.

Step D: Compound (I) Nebulization

8.27 g of Compound (I) from step C was dissolved in 60.77 g of ethylacetate by sonication/stirring (room temp.) to give a 12.00% (W/W)solution. This was fed at 5 ml/min to an industrial atomizer/nebulizer(Martin Walter Powersonic Model MW400GSIP, available from Sodeva,France), Power=70%, Amplitude=80%, Frequency=34.50 kHz and nebulizedinto 1.35 L of isopropyl alcohol (IPA) (20 times volumetric excesscompared to ethyl acetate volume) cooled to about −74±4° C. (coolingachieved via reactor jacket) and stirred at 200 rpm (to avoidmicrosphere agglomeration) in a jacketed reactor. De-ionised water at atemperature of 6° C. was fed at 1.5 L/min to the nebulizer jacket toeliminate any local heating effects which can cause fouling of thenebulizer tip due to ethyl acetate evaporation. The solution nebulizedevenly and an off-white particulate dispersion was seen to form in theIPA. This was allowed to thaw to about 0° C.–4° C. over a period ofabout 30 mins. to 2 hrs before passing it through a 125 μm sieve (toremove any large non-injectable droplets/particles) and on to a Whatmanno. 1 filter paper where it was vacuum-filtered. The filter cake wasrinsed with further IPA and then vacuum dried. 6.88 g of injectablematerial was obtained representing a yield of 83.19%. The microparticlesof Compound (I) had a mean particle size of about 54 microns.

The in vivo release of Compound (A) from microparticles of Compound (I)can be and were tested according to the following description. The invivo study was designed to evaluate the in vivo release profile ofCompound (A) following the intramuscular administration ofmicroparticles of Compound (I) to male Beagle dogs by means of thepharmacokinetic profile of Compound (A) following its administration.

Pharmaceutical formulations of microparticles of Compound (I) wereadministered intramuscularly in the rear leg muscles. Following a singleintramuscular administration of the irradiated or non-irradiatedprepared pharmaceutical forms containing microparticles of Compound (I)(amount of microparticles injected corresponded to 5 mg of Compound (A)based upon the determination that Compound (A) loading in Compound (I)was 11.23%) to groups of six dogs each per pharmaceutical form. Thequantitation of Compound (A) in serum samples and the pharmacokineticanalysis is conducted as follows.

Blood collection from the dogs are carried out before injection (time0), and 5, 15 and 30 min; 1, 2, 4, 8 and 12 hrs; 1, 2, 3 and 4 days;then twice per week over the first month (for example on Mondays andThursdays); and finally once a week from the second month untilcompleting the experiment, when the serum levels of Compound (A) were nolonger detected. However, the real times of blood collection wererecorded and used in the pharmacokinetic analysis. Blood samples (5 ml)at times 0 (before i.m. administration) and at 7, 21, 35, 56 and 84 daysafter the i.m. injection and blood samples (4 ml) for the rest ofsampling times, were taken through the jugular or the cephalic veins atthe prescribed times.

The samples were placed in two fractions: one about 2.5 ml or 3.5 ml incertain fixed time samplings, in tubes that contain 50 and 80 μl,respectively, of a solution of aprotinin (10 ml of Trasylol® 500000 KJUlypophillised and rediluted in 2 ml of p.p.i. water) and the other oneabout 1.5 ml in tubes that were allowed to stand.

After the red cells clot, the tubes were centrifuged for 20 min at 30000r.p.m. at +4° C. Serum with aprotinin were removed and stored in twofractions at −20° C. until the sample was analyzed for Compound (A).

The concentration of Compound (A) in the serum samples were analyzed bya radioimmunoassay method. Standard curves with blank dog plasma andCompound (A) standard solutions were prepared daily. In this method thelimit of quantification for Compound (A) in dog serum samples is about0.050 nanograms (ng)/ml. The areas under the curve (AUC) and the maximumserum concentration (C_(max)) were normalized by the dose supplied (doseadminstered to each of the animals expressed in μg/kg). The index of theabsorption rate (C_(max)/AUC) were also calculated.

The results of the foregoing experiment are shown in FIG. 1.

Compound (A) or a pharmaceutically-acceptable salt thereof, Compound (I)or microparticles of Compound (I) can be administered by oral,parenteral (e.g., intramuscular, intraperitoneal, intravenous orsubcutaneous injection, or implant), nasal, vaginal, rectal, sublingualor topical routes of administration and can be formulated withpharmaceutically acceptable carriers to provide dosage forms appropriatefor each route of administration.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, the activecompound is admixed with at least one inert pharmaceutically acceptablecarrier such as sucrose, lactose, or starch. Such dosage forms can alsocomprise, as is normal practice, additional substances other than suchinert diluents, e.g., lubricating agents such as magnesium stearate. Inthe case of capsules, tablets and pills, the dosage forms may alsocomprise buffering agents. Tablets and pills can additionally beprepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, the elixirscontaining inert diluents commonly used in the art, such as water.Besides such inert diluents, compositions can also include adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening, flavoring and perfuming agents.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, or emulsions. Examples ofnon-aqueous solvents or vehicles are propylene glycol, polyethyleneglycol, vegetable oils, such as olive oil and corn oil, gelatin, andinjectable organic esters such as ethyl oleate. Such dosage forms mayalso contain adjuvants such as preserving, wetting, emulsifying, anddispersing agents. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, by incorporating sterilizing agentsinto the compositions, by irradiating the compositions, or by heatingthe compositions. They can also be manufactured in the form of sterilesolid compositions which can be dissolved in sterile water, or someother sterile injectable medium immediately before use.

Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to the active substance,excipients such as coca buffer or a suppository wax.

Compositions for nasal or sublingual administration are also preparedwith standard excipients well known in the art.

It is preferred that the microparticles of Compound (I) be administeredvia parenteral administration or oral administration.

The effective dosage of the microparticles of Compound (I) to beadministered to a patient can be determined by the attending physicianor veterinarian and will be dependent upon the proper dosagescontemplated for Compound (A) and the loading of Compound (A) in themicroparticles of Compound (I). Such dosages will either be known or canbe determined by one of ordinary skill in the art. Preferably the dosageshould result in a level of at least 200 picograms/ml of Compound (A) inthe patient.

The use of immediate or of sustained release compositions depends on thetype of indications aimed at. If the indication consists of an acute orover-acute disorder, a treatment with an immediate release form will bepreferred over a prolonged release composition. On the contrary, forpreventive or long-term treatments, a prolonged release composition willgenerally be preferred.

Typically, the indication of upper gastrointestinal bleeding willcorrespond an acute or over-acute treatment with a dosage of about 80 to120 μg/day per person during approximately 5 days. After endoscopicaltreatment, preventive treatment against recurrence can be performedusing microparticles of Compound (A) or other sustained release forms asan adjuvant to usual treatments.

For other indications other than upper gastrointestinal bleeding, whichrequire rather long term treatments, microparticles of Compound (I) willbe preferred.

1. A composition comprising a complex comprised of Compound (A) havingthe formula

and a polymer wherein the polymer comprises 71% to 73% lactide units,26% to 28% glycolide units and 1% to 3% tartaric acid units and theamino group of Compound (A) is ionically bonded to a carboxylic group ofthe acid units of the polymer.
 2. A composition according to claim 1,wherein the polymer consists of 72% lactide units, 27% glycolide unitsand 1% tartaric acid units.
 3. A composition according to claim 2,wherein the percentage of Compound (A) in the composition is about 8% toabout 12%.
 4. A composition according to claim 1, wherein saidcomposition is in the form of microparticles.
 5. Microparticlesaccording to claim 4, wherein the mean microparticle size is about 10microns to about 100 microns.
 6. Microparticles according to claim 5,wherein the mean microparticle size is about 40 microns to about 70microns.
 7. Microparticles according to claim 6, wherein saidmicroparticles exhibit a zero-order release profile of Compound (A). 8.A pharmaceutical composition comprising microparticles of claim 4 and apharmaceutically acceptable carrier, diluent or adjuvant.